The PHYB-FOF2-VOZ2 module functions to fine-tune flowering in response to changes in light quality by modulating FLC expression in Arabidopsis.

Proper timing of flowering under different environmental conditions is critical for plant propagation. Light quality is a pivotal environmental cue that plays a critical role in flowering regulation. Plants tend to flower late under light with a high red (R)/far-red (FR) light ratio but early under light with a low R/FR light ratio. However, how plants fine-tune flowering in response to changes in light quality is not well understood. Here, we demonstrate that F-box of Flowering 2 (FOF2), an autonomous pathway-related regulator, physically interacts with VASCULAR PLANT ONE-ZINC FINGER 1 and 2 (VOZ1 and VOZ2), which are direct downstream factors of the R/FR light receptor phytochrome B (PHYB). We show that PHYB physically interacts with FOF2, mediates stabilization of the FOF2 protein under FR light and end-of-day FR light, and enhances FOF2 binding to VOZ2, which leads to degradation of VOZ2 by SCFFOF2 E3 ligase. By contrast, PHYB mediates degradation of FOF2 protein under R light and end-of-day R light. Genetic interaction studies demonstrated that FOF2 functions downstream of PHYB to promote FLC expression and inhibit flowering under both high R/FR light and simulated shade conditions, processes that are partially dependent on VOZ proteins. Taken together, our findings suggest a novel mechanism whereby plants fine-tune flowering time through a PHYB-FOF2-VOZ2 module that modulates FLC expression in response to changes in light quality.

Molecular mechanism governing RNA-binding property of mammalian TRIM71 protein.

TRIM71 is an RNA-binding protein with ubiquitin ligase activity. Numerous functions of mammalian TRIM71, including cell cycle regulation, embryonic stem cell (ESC) self-renewal, and reprogramming of pluripotent stem cells, are related to its RNA-binding property. We previously reported that a long noncoding RNA (lncRNA) Trincr1 interacts with mouse TRIM71 (mTRIM71) to repress FGF/ERK pathway in mouse ESCs (mESCs). Herein, we identify an RNA motif specifically recognized by mTRIM71 from Trincr1 RNA, and solve the crystal structure of the NHL domain of mTRIM71 complexed with the RNA motif. Similar to the zebrafish TRIM71, mTRIM71 binds to a stem-loop structured RNA fragment of Trincr1, and an adenosine base at the loop region is crucial for the mTRIM71 interaction. We map similar hairpin RNAs preferably bound by TRIM71 in the mRNA UTRs of the cell-cycle related genes regulated by TRIM71. Furthermore, we identify key residues of mTRIM71, conserved among mammalian TRIM71 proteins, required for the RNA-binding property. Single-site mutations of these residues significantly impair the binding of TRIM71 to hairpin RNAs in vitro and to mRNAs of Cdkn1a/p21 and Rbl2/p130 in mESCs. Furthermore, congenital hydrocephalus (CH) specific mutation of mTRIM71 impair its binding to the RNA targets as well. These results reveal molecular mechanism behind the recognition of RNA by mammalian TRIM71 and provide insights into TRIM71 related diseases.

Photosynthetic characteristics and genetic mapping of a new yellow leaf mutant crm1 in Brassica napus.

Chlorophyll is one of the key factors for photosynthesis and plays an important role in plant growth and development. We previously isolated an EMS mutagenized rapeseed chlorophyll-reduced mutant (crm1), which had yellow leaf, reduced chlorophyll content and fewer thylakoid stacks. Here, we found that crm1 showed attenuated utilization efficiency of both light energy and CO2 but enhanced heat dissipation efficiency and greater tolerance to high-light intensity. BSA-Seq analysis identified a single nucleotide change (C to T) and (G to A) in the third exon of the BnaA01G0094500ZS and BnaC01G0116100ZS, respectively. These two genes encode the magnesium chelatase subunit I 1 (CHLI1) that catalyzes the insertion of magnesium into protoporphyrin IX, a pivotal step in chlorophyll synthesis. The mutation sites resulted in an amino acid substitution P144S and G128E within the AAA+ domain of the CHLI1 protein. Two KASP markers were developed and co-segregated with the yellow leaf phenotype in segregating F2 population. Loss of BnaA01.CHLI1 and BnaC01.CHLI1 by CRISPR/Cas9 gene editing recapitulated the mutant phenotype. BnaA01.CHLI1 and BnaC01.CHLI1 were located in chloroplast and highly expressed in the leaves. Furthermore, RNA-seq analyses revealed the expression of chlorophyll synthesis-related genes were upregulated in the crm1 mutant. These findings provide a new insight into the regulatory mechanism of chlorophyll synthesis in rapeseed and suggest a novel target for improving the photosynthetic efficiency and tolerance to high-light intensity in crops. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01429-6.

KLF17 promotes human naive pluripotency through repressing MAPK3 and ZIC2.

The pluripotent state of embryonic stem cells (ESCs) is regulated by a sophisticated network of transcription factors. High expression of KLF17 has recently been identified as a hallmark of naive state of human ESCs (hESCs). However, the functional role of KLF17 in naive state is not clear. Here, by employing various gain and loss-of-function approaches, we demonstrate that KLF17 is essential for the maintenance of naive state and promotes the primed to naive state transition in hESCs. Mechanistically, we identify MAPK3 and ZIC2 as two direct targets repressed by KLF17. Overexpression of MAPK3 or ZIC2 partially blocks KLF17 from promoting the naive pluripotency. Furthermore, we find that human and mouse homologs of KLF17 retain conserved functions in promoting naive pluripotency of both species. Finally, we show that Klf17 may be essential for early embryo development in mouse. These findings demonstrate the important and conserved function of KLF17 in promoting naive pluripotency and reveal two essential transcriptional targets of KLF17 that underlie its function.

Rational construction of porous N-doped Fe2O3 films on porous graphene foams by molecular layer deposition for tunable microwave absorption.

Graphene-based materials with porous microstructure have attracted immense attentions due to their wide application in microwave absorption. However, constructing magnetic film with both porous microstructure and uniform pore size by using traditional methods still remains a challenge. To overcome this problem, we reported a facile strategy of molecular layer deposition (MLD) for successfully fabrication of the hybrid-architecture of porous graphene foams and nitrogen-doped porous Fe2O3 films. The surfaces of porous graphene foams are uniformly covered by porous Fe2O3 films without aggregation and the pore structures are widely distributed. The porous graphene-based composites exhibit remarkably enhanced microwave absorption performance compared to the pristine graphene foams. The minimum reflection loss value is increased by approximately 8 times, reaching -64.36 dB with a thickness of only 2.18 mm. More importantly, the absorption property can be precisely modulated by tuning the MLD cycle numbers and effective absorption bandwidth covers 3.04-18.0 GHz by adjusting the thickness from 1.0 to 5.0 mm. This work provides new insights for exploring novel and high-performance graphene-based microwave absorbents and offers a new idea to rationally design three-dimensional composites with porous magnetic films.

Enhanced therapeutic efficacy of Listeria-based cancer vaccine with codon-optimized HPV16 E7.

Cervical cancer is a leading cause of high mortality in women in developing countries and has a serious impact on women's health. Human papilloma virus (HPV) prophylactic vaccines have been produced and may hold promise for reducing the incidence of cervical cancer. However, the limitations of current HPV vaccine strategies make the development of HPV therapeutic vaccines particularly important for the treatment of HPV related lesions. Our previous work has demonstrated that LM4Δhly::E7 was safe and effective in inducing antitumor effect by antigen-specific cellular immune responses and direct killing of tumor cell on a cervical cancer model. In this study, the codon usage effect of a novel Listeria-based cervical cancer vaccine LM4Δhly::E7-1, was evaluated for effects of codon-optimized E7 expression, cellular immune response and therapeutic efficacy in a tumor-bearing murine model. Our data demonstrated that up-regulated expression of E7 was strikingly elevated by codon usage optimization, and thus induced significantly higher Th1-biased immunity, lymphocyte proliferation, and strong specific CTL activity ex-vivo compared with LM4Δhly::E7-treated mice. Furthermore, LM4Δhly::E7-1 enhanced a remarkable therapeutic effect in establishing tumors. Taken together, our results suggest that codon usage optimization is an important consideration in constructing live bacterial-vectored vaccines and is required for promoting effective T cell responses.

Acidic pH transiently prevents the silencing of self-renewal and dampens microRNA function in embryonic stem cells.

Enhanced glycolysis is a distinct feature associated with numerous stem cells and cancer cells. However, little is known about its regulatory roles in gene expression and cell fate determination. Here, we confirm that glycolytic metabolism and lactate production decrease during the differentiation of mouse embryonic stem cells (mESCs). Importantly, acidic pH due to lactate accumulation can transiently prevent the silencing of mESC self-renewal in differentiation conditions. Furthermore, acidic pH partially blocks the differentiation of human ESCs (hESCs). Mechanistically, acidic pH downregulates AGO1 protein and de-represses a subset of mRNA targets of miR-290/302 family of microRNAs which facilitate the exit of naive pluripotency state in mESCs. Interestingly, AGO1 protein is also downregulated by acidic pH in cancer cells. Altogether, this study provides insights into the potential function and underlying mechanism of acidic pH in pluripotent stem cells (PSCs).

Icariin induces apoptosis by suppressing autophagy in tamoxifen-resistant breast cancer cell line MCF-7/TAM.

BACKGROUND: Icariin is a major component isolated from Epimedium brevicornum Maxim and has been reported to exhibit anti-tumor activity. However, whether icariin could reverse the acquired drug resistance in breast cancer remains largely unclear. Therefore, this study was designed to explore the antitumor effects of icariin and its underlying mechanisms in a tamoxifen-resistant breast cancer cell line MCF-7/TAM. METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Lactate dehydrogenase (LDH) assay were performed to determine the effects of icariin on cell viability and cell death. Cell cycle progression and apoptosis were detected by flow cytometry analysis. Transmission electron microscopy (TEM) assay was utilized to observe cell autophagy. The downstream protein levels were measured using western blotting. RESULTS: Here, we observed that icariin treatment not only inhibited the growth of MCF-7 but also has a potential function to overcome tamoxifen resistance in MCF-7/TAM. Moreover, icariin significantly induced cell cycle G0/G1 phase arrest and apoptosis, as well as suppressed autophagy. At molecular levels, icariin treatment remarkably down-regulated the expression levels of CDK2, CDK4, Cyclin D1, Bcl-2, LC3-1, LC3-II, AGT5, Beclin-1, but upregulated the expression levels of caspase-3, PARP and p62. Most importantly, we found inhibition of autophagy via 3-MA treatment could significantly enhance the effects of icariin on cell viability and apoptosis. Enhanced autophagy via autophagy related 5 (ATG5) overexpression could partially reverse the effects of icariin on cell viability and apoptosis. CONCLUSION: These results revealed that icariin might potentially be useful as an adjuvant agent in cancer chemotherapy to enhance the effect of tamoxifen through suppression of autophagy in vitro and provide insight into the therapeutic potential of icariin for the treatment of chemo-resistant breast cancer.

A TRIM71 binding long noncoding RNA Trincr1 represses FGF/ERK signaling in embryonic stem cells.

Long noncoding RNAs (lncRNAs) have emerged as important components of gene regulatory network in embryonic stem cells (ESCs). However, the function and molecular mechanism of lncRNAs are still largely unknown. Here we identifies Trincr1 (TRIM71 interacting long noncoding RNA 1) lncRNA that regulates the FGF/ERK signaling and self-renewal of ESCs. Trincr1 is exported by THOC complex to cytoplasm where it binds and represses TRIM71, leading to the downregulation of SHCBP1 protein. Knocking out Trincr1 leads to the upregulation of phosphorylated ERK and ERK pathway target genes and the decrease of ESC self-renewal, while knocking down Trim71 completely rescues the defects of Trincr1 knockout. Furthermore, ectopic expression of Trincr1 represses FGF/ERK signaling and the self-renewal of neural progenitor cells (NPCs). Together, this study highlights lncRNA as an important player in cell signaling network to coordinate cell fate specification.

Fast and simple transesterification of epoxidized soybean oil to prepare epoxy methyl esters at room temperature.

Epoxidized soybean oil methyl esters were prepared via transesterification of epoxidized soybean oil (ESBO) with methanol catalyzed by cheap and stable sodium hydroxide (NaOH). The transesterification could be completed in only 5 min at room temperature (25 °C) without loss of the epoxide function and the transesterification rate was promoted significantly while the utilization of 5% acetone as co-solvent. The afforded products epoxidized methyl esters represent a renewable substrate that is readily converted into surfactants, fuel additives and other valuable industrial products.

MicroRNAs and RNA binding protein regulators of microRNAs in the control of pluripotency and reprogramming.

Post-transcriptional and translational regulations play essential roles during cellular reprogramming and in the maintenance and differentiation of pluripotent stem cells (PSCs). MicroRNAs (miRNAs) control cell cycle, glycolysis, chromatin state, survival and pluripotency of ESCs. Likewise, many miRNAs assist or act as a barrier for the generation of induced pluripotent stem cells (iPSCs). Recent studies also reveal exciting new directions on miRNA functions in regulating the switch between naive and primed pluripotent states as well as the establishment of totipotent-like state. Furthermore, the biogenesis and function of pluripotency related miRNAs are regulated by various RNA binding proteins (RBPs) at different levels. Revealing the interplay between RBPs and miRNAs will advance our understanding of molecular mechanisms controlling pluripotency and provide better means to manipulate PSCs for clinical applications. In this review, we summarize recent findings on the function of miRNAs in ESCs and during reprogramming. In addition, we also discuss new directions on miRNA functions in regulating the switch between different pluripotent states and RBP-mediated regulation of miRNA biogenesis and function in pluripotency control.

Pluripotency-associated miR-290/302 family of microRNAs promote the dismantling of naive pluripotency.

The molecular mechanism controlling the dismantling of naive pluripotency is poorly understood. Here we show that microRNAs (miRNAs) have important roles during naive to primed pluripotency transition. Dgcr8(-/-) embryonic stem cells (ESCs) failed to completely silence the naive pluripotency program, as well as to establish the primed pluripotency program during differentiation. miRNA profiling revealed that expression levels of a large number of miRNAs changed dynamically and rapidly during naive to primed pluripotency transition. Furthermore, a miRNA screen identified numerous miRNAs promoting naive to primed pluripotency transition. Unexpectedly, multiple miRNAs from miR-290 and miR-302 clusters, previously shown as pluripotency-promoting miRNAs, demonstrated the strongest effects in silencing naive pluripotency. Knockout of both miR-290 and miR-302 clusters but not either alone blocked the silencing of naive pluripotency program. Mechanistically, the miR-290/302 family of miRNAs may facilitate the exit of naive pluripotency in part by promoting the activity of MEK pathway and through directly repressing Akt1. Our study reveals miRNAs as an important class of regulators potentiating ESCs to transition from naive to primed pluripotency, and uncovers context-dependent functions of the miR-290/302 family of miRNAs at different developmental stages.

[Construction and characterization of an attenuated recombinant Listeria monocytogenes vector vaccine delivering HPV16 E7].

Listeria monocytogenes (L. monocytogenes, LM) is an excellent tumor vaccine vector. In this study, recombinant LM vaccine candidate expressing human papillomavirus type 16 (HPV16) E7 protein was constructed and its charactericts were determined. Through homologous recombination, E7 gene was cloned in frame with the LM4 Phly promoter-signal sequence, and introduced into the chromosome of LM4. The recombinant strain named LM4△hly::E7 with the plasmid-free and antibiotic-resistant gene-free was constructed. LM4△hly::E7 could express and secrete E7-LLO fusion protein; its size is 66 kDa and has immunological activity. Furthermore, LM4△hly::E7 could multiply in RAW264.7 macrophages by confocal laser scanning microscope. Additionally, LM4△hly::E7 could induce specific antibodies against E7 in immunized mice in ELISA. Also, the 50% lethal dose (LD50) of LM4△hly::E7 strain was 3.863×109 CFU (Colony-Forming Units) in C57BL/6 mice with intraperitoneal immunization, which was more attenuated than wild type LM4. Mice immunized with LM4△hly::E7 did not show obvious pathological change. These data show that LM4△hly::E7 expressing E7-LLO fusion protein has good safety, which may provide the materials for research of antitumor effect and would be a promising vaccine candidate for cervical cancer.

[Construction and characterization of Listeria monocytogenes deltahfq mutant strain].

OBJECTIVE: Gram-positive brevibacterium Listeria monocytogenes (Lm) is an important zoonotic foodborne pathogen, engaged in both saprophytism and parasitism. It could adapt, survive and display pathogenicity under different environmental stress challenges, which is associated with the regulatory network consisting of regulating factors. The biological characterizations of regulator hfq was evaluated in this study. METHODS: hfq deleted serovar 1/2 a strain EGDe was constructed with homologous recombination, the biological characteristics of the mutant strain was compared with its parental strain. RESULTS: The growth of EGDe delta hfq was significantly inhibited under cold temperature (P < 0.05), salt medium containing 7% NaCl and the medium containing 4.5% ethanol. The ability of biofilm formation of the mutant strain in BactoTM Brain Heart Infusion (BHI) was reduced significantly (P < 0.05); notably, the invasion rate to Caco-2 cell lines was obviously reduced. Infection capacity of EGDe delta hfq to BALB /c mice decreased and the LDD was 6 times higher than EGDe. CONCLUSION: Hfq protein of Listeria monocytogenes plays an important role in regulating bacterial virulence, biofilm formation and stress response. This deletion strain provided material to further study the function of Hfq and provides the possibilities to elucidate the mechanisms of Lm in resisting the stress and paves ways to the development of novel strategies for the prevention and control of Lm infections.

Conservation of imprinting of Neuronatin (Nnat) in rabbits.

Although the expression and epigenetic status of imprinted genes have been extensively studied in a number of species, less is known about the genomic imprinting in rabbits. Neuronatin (Nnat) plays significant roles in the brain development and metabolic regulation and has been identified to be imprinted and paternally expressed in humans, mice and pigs; however, it has not yet been investigated in rabbits. In this study, we confirmed the expression of two isoforms of the rabbit Nnat (Nnat-a and Nnat-ß) identified in Genbank and Ensembl by quantitative real-time PCR. In addition, we also determined the methylation profile of the CpG island in the promoter region of the rabbit Nnat using bisulfite sequencing PCR and combined bisulfite restriction analysis. Here, we provide the first evidence that Nnat has two transcripts in rabbit. Additionally, the CpG island located in the promoter region shows oocyte-specific methylation and may be the differentially methylated region of Nnat in rabbits.

NiO/nanoporous graphene composites with excellent supercapacitive performance produced by atomic layer deposition.

Nickel oxide (NiO) is a promising electrode material for supercapacitors because of its low cost and high theoretical specific capacitance of 2573 F g(-1). However, the low electronic conductivity and poor cycling stability of NiO limit its practical applications. To overcome these limitations, an efficient atomic layer deposition (ALD) method is demonstrated here for the fabrication of NiO/nanoporous graphene (NG) composites as electrode materials for supercapacitors. ALD allows uniform deposition of NiO nanoparticles with controlled sizes on the surface of NG, thus offering a novel route to design NiO/NG composites for supercapacitor applications with high surface areas and greatly improved electrical conductivity and cycle stability. Electrochemical measurements reveal that the NiO/NG composites obtained by ALD exhibited excellent specific capacitance of up to ∼ 1005.8 F g(-1) per mass of the composite electrode (the specific capacitance value is up to ∼ 1897.1 F g(-1) based on the active mass of NiO), and stable performance after 1500 cycles. Furthermore, electrochemical performance of the NiO/NG composites is found to strongly depend on the size of NiO nanoparticles.

Aberrant expression of Xist in aborted porcine fetuses derived from somatic cell nuclear transfer embryos.

Cloned pigs generated by somatic cell nuclear transfer (SCNT) show a greater ratio of early abortion during mid-gestation than normal controls. X-linked genes have been demonstrated to be important for the development of cloned embryos. To determine the relationship between the expression of X-linked genes and abortion of cloned porcine fetuses, the expression of X-linked genes were investigated by quantitative real-time polymerase chain reaction (q-PCR) and the methylation status of Xist DMR was performed by bisulfate-specific PCR (BSP). q-PCR analysis indicated that there was aberrant expression of X-linked genes, especially the upregulated expression of Xist in both female and male aborted fetuses compared to control fetuses. Results of BSP suggested that hypomethylation of Xist occurred in aborted fetuses, whether male or female. These results suggest that the abnormal expression of Xist may be associated with the abortion of fetuses derived from somatic cell nuclear transfer embryos.

Altered imprinted gene expression and methylation patterns in mid-gestation aborted cloned porcine fetuses and placentas.

PURPOSE: To determine the expression patterns of imprinted genes and their methylation status in aborted cloned porcine fetuses and placentas. METHODS: RNA and DNA were prepared from fetuses and placentas that were produced by SCNT and controls from artificial insemination. The expression of 18 imprinted genes was determined by quantitative real-time PCR (q-PCR). Bisulfite sequencing PCR (BSP) was conducted to determine the methylation status of PRE-1 short interspersed repetitive element (SINE), satellite DNA and H19 differentially methylated region 3 (DMR3). RESULTS: The weight, imprinted gene expression and genome-wide DNA methylation patterns were compared between the mid-gestation aborted and normal control samples. The results showed hypermethylation of PRE-1 and satellite sequences, the aberrant expression of imprinted genes, and the hypomethylation of H19 DMR3 occurred in mid-gestation aborted fetuses and placentas. CONCLUSIONS: Cloned pigs generated by somatic cell nuclear transfer (SCNT) showed a greater ratio of early abortion during mid-gestation than did normal controls because of the incomplete epigenetic reprogramming of the donor cells. Altered expression of imprinted genes and the hypermethylation profile of the repetitive regions (PRE-1 and satellite DNA) may be associated with defective development and early abortion of cloned pigs, emphasizing the importance of epigenetics during pregnancy and implications thereof for patient-specific embryonic stem cells for human therapeutic cloning and improvement of human assisted reproduction.

Genomic imprinting analysis of Igf2/H19 in porcine cloned fetuses using parthenogenetic somatic cells as nuclear donors.

To gain insight into parthenogenesis in pigs, we report for the first time that using parthenogenetic somatic cells as nuclear donors (PSCNT), the porcine parthenogenetic fetus can develop to gestational day 39. Weight and morphological analysis revealed that PSCNT fetuses were smaller and developmentally retarded when compared to normally fertilized controls. Quantitative gene expression analysis indicated that in PSCNT fetuses, H19 was over-expressed, whereas Igf2 was significantly reduced (p < 0.05) compared with their controls. In addition, bisulfite-sequencing PCR results demonstrated that H19 differentially DNA methylated regions (DMRs) were hypomethylated in PSCNT fetuses, while Igf2 DMRs were hypermethylated in both PSCNT and control fetuses. Our results suggest that extended development of the porcine parthenogenetic fetus can be accomplished using PSCNT and that abnormal DNA methylation of H19 DMRs might contribute to the critical barrier of parthenogenesis in pigs.

Leukemoid reaction in sarcomatoid renal cell carcinoma: a two-case report.

Leukemoid reaction is defined as reactive leukocytosis exceeding 40 × 109/l, with a significant increase in early neutrophil precursors, and can be a paraneoplastic manifestation of various malignant tumors. Leukemoid reaction is a sign for poor prognosis in solid tumors so is sarcomatoid renal cell carcinoma (SRCC) when compared to more differentiated histologies. Here, we are reporting two cases of leukemoid reaction after radical nephrectomy, both of which were diagnosed as SRCC pathologically. The operations were successful: no complications were observed and the patients were discharged in good condition. However, a few weeks later, the white blood cell (WBC) count gradually increased. Even though routine management was done immediately, the count was still elevating. A diagnosis of a leukemoid reaction was established and both of them died shortly thereafter. Due to the poor prognosis of most patients with malignant leukemoid reaction, leukemoid reaction may be a predictor of prognosis in patients with SRCC, but more data are needed.